Convenient chromatographic supports which are stable, have high capacity, and have low nonspecific adsorption, have long been sought. However, this combination of properties is singularly difficult to achieve. Single-substance supports such as charcoal or synthetic polymers are nonspecific, and apparently cannot be made with both high capacity and stability. Hybrid gels, such as that obtained by impregnating porous silica with DEAE dextran, also suffer these defects.
A number of hybrid supports of this nature have been disclosed, for example, in U.S. Pat. No. 4,673,734, directed to a mineral support impregnated with aminated polysaccharide; U.S. Pat. No. 3,577,226, describing polymerization and formation of a cross-linked polymer in the pores of silica gel in situ: Boardman, N. K., J Chromatog (1959) 2:388-389, which describes the formation of a thin layer of resin in the cavity of a porous support such as celite; and U.S. Pat. No. 3,878,092, which also describes a polymeric-coated silica.
Uncoated forms of silica and porous glass, while providing a high porosity and flow rate, are susceptible to degradation and nonspecific adsorption of proteins because of silanol groups at the surface. In order to overcome these disadvantages, hydrophilic polymeric coatings involving silane coupling agents have also been disclosed. For example, U.S. Pat. Nos. 3,983,299 and 4,029,583 describe glycidoxypropyl trimethoxysilane attached to a silica support. However, the adhesion qualities of the coating are poor.
U.S. Pat. No. 4,332,694 describes the combination of a reactive epoxy with an inorganic silica support. U.S. Pat. No. 4,352,884 describes coating of inorganic materials with a copolymer of hydrophilic acrylate or methacrylate along with a copolymerizable carboxylic acid or amine, and a cross-linking agent, a procedure which resulted in insufficient binding to the underlying substrate. U.S. Pat. No. 3,795,313 describes a siliceous support coated with a methacryloxysilane; U.S. Pat. No. 3,808,125 describes a silica support chemically bonded to a copolymer made from a coupling agent polymerized onto a polymeric backbone. In a different approach, U.S. Pat. No. 4,070,348 describes copolymers of glycidyl and amino-containing acrylates which are covalently modified with specific ligands, such as enzymes or proteins.
European patent application No. 0172579, published 26 Feb. 1986 and the U.S. Pat. No. 4,724,207, describe a modified silica support covalently bonded to a synthetic copolymer which contains a polymer which can be covalently coupled directly to the silica copolymerized with a material which contains either an ionizable group, a hydrophobic material, or a group capable of binding an affinity ligand. A related U.S. Pat. No. 4,663,163 describes and claims similarly modified polysaccharide supports. Thus, these supports use a subsequently cross-linked polymeric coating as a matrix to contain the specificity-conferring derivatization and as a link to bind this material to the particulate organic or inorganic support.
None of the foregoing-described chromatographic supports would be suitable for use in hemoperfusion, either because the flow properties are inadequate, because the supports are too unstable, or because nonspecific binding is too prevalent. The foregoing approaches may also result in hydrogels associated with the acrylic polymers which are inherently disadvantageous as poorly adhering and unduly significant in modifying the mechanical properties of the basic particles. For example, polyacrylic hydrogels have calculated average pore radii of only 4-10 angstroms (Refojo, M. F., J Ap Polym Sci (1965) 9:3417; White, M. L., J Phys Chem (1960) 64:1563. Such pore sizes effectively exclude even small plasma proteins such as albumin (158 .ANG..times.38 .ANG.) and gamma globulin (235 .ANG..times.44 A.ANG.).
For the foregoing reasons, the above-referenced chromatographic supports are inappropriate for ex vivo treatment of biological fluids such as blood or plasma. Suitability for such use requires high dimensional stability, without any particulate release, high efficiency and capacity, and biocompatibility, including lack of nonspecific adsorption. In presently practiced techniques, nonspecific adsorbents, such as activated charcoal, ion exchangers or resins, have been used for plasma perfusion, which is easier to conduct than hemoperfusion but requires additional equipment to separate cells from plasma and may also involve filtration of treated plasma. Attempts to perform specific removal of blood components have been reported, such as the passage of blood through a tube coated with a specific immunoligand (Schenkein et al, J Clin Invest (1971) 50:1964; Lyle et al, J Immunol (1974) 113:517). Terman et al, Clin Exp Immunol (1977) 28:180, describes an encapsulated sorbent coupled to nylon and used as column, Terman et al, New Eng J Med (1981) 305:1195-1200, describes the use of protein A bound to charcoal-collodion to treat solid tumors, and Besa et al, Am J Med (1981) 71:1035, describes a stabilized protein A to remove serum IgG in an autoimmune therapy. U.S. Pat. No. 4,681,870 discloses the use of a protein A silica immunosorbent to remove IgG from biological fluids, a process which suffers from the disadvantage of the release of "fines" during the ex vivo treatment. Messaikeh et al, "Biological and Biomechanical Performance of Biomaterials" (1986), Christel et al, eds, Elsevier, Amsterdam, pp. 321-326, describes use of derivatives of polystyrene to remove Factor VIII:C ex vivo. Margel et al, Ap Biochem Biotechnol (1986) 12:37-66, describes the use of derivatized cross-linked agarose polyacrolein microspheric beads ("agarose acrobeads") for specific hemoperfusion.
The use of specific affinity ligands coupled directly to an inorganic support as a matrix for selective removal of materials from the plasma or blood was described by Bensinger et al in a series of articles appearing in Transfusion (1981) 21:335-342; New Eng J Med (1981) 304:160-162; J Clin Apheresis (1982) 1:2-5; and Vox Sang (1985) 48:357-361. In the latest of these disclosures, the immunoadsorbent was thinly coated with collodion applied by the method described by Chang, Trans Am Soc Artif Int Organs (1980) 26:546-549 and the related U.S. Pat. No. 3,725,113, but the thin collodion coating did not prevent the release of fines. Hydrophilic coatings of nonspecific supports are described. Others have used similar columns for removal of antibodies from human plasma (Osterwalder et al, Blut (1986) 53:379-390; Bussel et al, Plasma Ther Transfus Technol (1985) 6:461-464) and from whole blood (Raja et al, ibid (1986) 22:102-103, and Bannett et al, Transplantation (1987) 43:909-910). Attempts to coat adsorbents also include the use of glow discharge to polymerize hexamethyldisiloxane on the surface of activated charcoal granule for hemoperfusion (Hasirci and Akovali, J Biomed Mater Res (1986) 20:963-970), again failing to prevent the release of fines.
An additional problem is non-specific adsorption to the support. The adhesion of various materials to polymeric substances has been studied. A review of biocompatibility of various polymers is found in Neumann et al, in "Biocompatible Polymers, Metals and Composites" (1983) (Szycher, ed), Technomic, Pa. For example, polystyrene has been shown to be a poor adherent for cells (Thomas et al, in "Biological and Biomechanical Performance of Biomaterials" (supra)). Platelet adhesion does not seem to depend on surface smoothness or roughness (Zingg et al, Biomaterials (1981) 2:156-158); however, for hydrophobic surfaces surface roughness does affect cell adhesion under flow conditions (Strong et al, Anal Biomed Engg (1982) 10:71-82).
The present invention provides a process for providing a controlled pore coating with membrane-type physical properties conferring integrity and mechanical strength, which is biocompatible for use in protecting affinity supports to prevent the release of fines. The coating is thus consistent with suitable mechanical properties of membranes, and is of appropriate porosity to accommodate the penetration of blood proteins such as antibodies or other materials for which an affinity ligand attached to the support is reactive.